Bone mineral density in adults with arthrogryposis multiplex congenita: a retrospective cohort analysis

The primary objective of this study was to evaluate the prevalence of low femoral and lumbar spine bone mineral density (BMD) in adults with arthrogryposis multiplex congenita (AMC). We performed a retrospective cohort analysis of adults with AMC who were enrolled in the French Reference Center for AMC and in the Pediatric and Adult Registry for Arthrogryposis (PARART, NCT05673265). Patients who had undergone dual-energy X-ray absorptiometry (DXA) and/or vitamin D testing were included in the analysis. Fifty-one patients (mean age, 32.9 ± 12.6 years) were included; 46 had undergone DXA. Thirty-two (32/51, 62.7%) patients had Amyoplasia, and 19 (19/51, 37.3%) had other types of AMC (18 distal arthrogryposis, 1 Larsen). Six patients (6/42, 14.3%) had a lumbar BMD Z score less than − 2. The mean lumbar spine Z score (− 0.03 ± 1.6) was not significantly lower than the expected BMD Z score in the general population. Nine (9/40, 22.5%) and 10 (10/40, 25.0%) patients had femoral neck and total hip BMD Z scores less than − 2, respectively. The mean femoral neck (− 1.1 ± 1.1) and total hip (− 1.2 ± 1.2) BMD Z scores in patients with AMC were significantly lower than expected in the general population (p < 0.001). Femoral neck BMD correlated with height (rs = 0.39, p = 0.01), age (rs = − 0.315, p = 0.48); total hip BMD correlated with height (rs = 0.331, p = 0.04) and calcium levels (rs = 0.41, p = 0.04). Twenty-five patients (25/51, 49.0%) reported 39 fractures. Thirty-one (31/36, 86.1%) patients had 25-hydroxyvitamin D levels less than 75 nmol/l, and 6 (6/36, 16.7%) had 25-hydroxyvitamin D levels less than 75 nmol/l. Adults with AMC had lower hip BMD than expected for their age, and they more frequently showed vitamin D insufficiency. Screening for low BMD by DXA and adding vitamin D supplementation when vitamin D status is insufficient should be considered in adults with AMC, especially if there is a history of falls or fractures.


DXA analysis
Lumbar spine (L1-L4), femoral neck and total hip BMD were measured by DXA in the University Hospital Grenoble-Alpes Radiology Department, which specializes in osteoarticular disease.All BMD values were obtained on the same densitometer (Lunar iDXA, GE Healthcare).Lumbar spine, right and left femoral neck and total hip BMD (g/cm 2 ) were transformed to age-and sex-specific Z scores according to the manufacturer's reference data.As our population was predominantly young (premenopausal women and men under 50 years), we chose to use the Z score, in accordance with the 2023 Official Positions for Adults of the International Society of Clinical Densitometry (ISCD) 15 .A Z score of − 2.0 or less indicated that the BMD was below the expected range for individuals of that age.The Z score is the number of standard deviations from the mean BMD of a healthy population of the same age and sex.Quality control for instrumentation was performed daily, using a spine phantom provided by the manufacturer, prior to any measurement.Lumbar spine, femoral neck and total hip BMD and Z score values were collected from the patients' medical records by one of the investigators (XR).The quality of BMD analysis was graded by two independent investigators (XR, RG) as perfect, not perfect but interpretable, or uninterpretable.If the quality score was equivalent between the left and right femurs, the side with the lower femoral neck BMD and Z score was selected.Lumbar and femoral BMD and Z scores from DXA analyses considered uninterpretable were excluded from the statistical analysis.
Calcium and phosphate levels were measured via a colorimetric assay (Vista -Siemens).Calcium levels < 2.12 mmol/l and > 2.60 mmol/l indicated hypocalcemia and hypercalcemia, respectively.Phosphate levels < 0.80 mmol/l and > 1.45 mmol/l indicated hypophosphatemia and hyperphosphatemia, respectively.Calcium, phosphate and 25-OHD levels were collected from the patients' medical records by one of the investigators (XR).

Statistical analysis
Mann-Whitney tests and t tests were used to compare two groups with nonnormally and normally distributed data, respectively.The Shapiro-Wilk test was used to test for a normal distribution.Variables are expressed as the mean ± standard deviation (SD) or median [first quartile (Q1)-third quartile (Q3)].
For categorical data, the chi-square test or Fisher's exact test was used to compare two groups.Categorical data are expressed as numbers and percentages.
To test whether the mean Z scores were significantly different from zero (i.e. the mean result expected in the general population), we used a one-sample t test.Correlations between lumbar spine, femoral neck and total hip BMD and age, height, weight, body mass index (BMI), calcium level, phosphate level, 25-OHD level, 6MWT distance and total FIM score were assessed via Spearman rank correlation tests (r s ).Age, sex, type of AMC, height, weight, BMI, history of fracture, calcium level, phosphate level, 25-OHD, 6MWT distance, total FIM score and limited ambulation were evaluated as potential factors associated with lumbar or femoral neck BMD Z scores lower than − 2 using univariate logistic regression analysis.Patients with missing data were excluded from the statistical analysis; missing data are reported in the Results tables.A p value < 0.05 was considered to indicate significance.Statistical analyses were performed using Jamovi (version 1.6.23)and R + + software (version 1.5.07).

Lumbar spine, femoral neck and total hip BMD
Forty-six (46/56, 82.1%) patients had undergone DXA at Grenoble Alpes University Hospital.There were no significant differences between patients who did not undergo DXA and patients who did undergo DXA (data not shown).Four patients had uninterpretable lumbar spine BMD results due to significant scoliosis or spinal fixation devices.Eighteen patients had uninterpretable femoral neck results, including 4 patients whose femoral neck BMD was uninterpretable on both sides because the region of interest could not be correctly positioned due to joint contractures (Fig. 2) and 2 patients whose femoral neck BMD was unavailable.None of the patients who underwent DXA had uninterpretable BMD results at all three sites (lumbar spine, right hip and left hip).Lumbar spine DXA analyses revealed a mean BMD of 1.14 (± 0.21) g/cm 2 ; 22 patients (22/42, 52.4%) had Z scores less than 0, and 6 patients (6/42, 14.3%) had Z scores less than -2.The mean BMD Z score for the lumbar spine was not significantly lower than zero.Individuals with other types of AMC had significantly lower Z scores than those with Amyoplasia (Table 2).The mean BMD Z score for the lumbar spine was not significantly lower than zero for patients with Amyoplasia or other types of AMC.

Fracture history and BMD
Twenty-five patients (25/51, 49.0%) reported 39 fractures (an average of 1.6 fractures per patient).Twenty-two (22/39, 56.4%) of the fractures involved the upper limb (7 humerus, 5 forearm, 3 elbow, 1 acromion, 1 scaphoid, 1 scapula and 4 unspecified locations), 41.0% (16/39) involved the lower limb (8 femur, 4 tibia, 1 calcaneum, 1 foot and 2 unspecified locations), and 2.6% (1/39) involved the pelvis.No cases of vertebral fracture were reported.A total of 42.1% (16/38) of fractures occurred after the age of 16.The proportion of fractures caused by low-level trauma equivalent to a fall from standing height or less could not be determined due to the lack of precise data in the medical records.The lumbar spine, femoral neck and total hip BMD and Z score did not differ according to the history of fracture.

Discussion
The present study showed for the first time that adults with AMC had low femoral neck BMD and a higher frequency of vitamin D deficiency, whereas the BMD of the lumbar spine was not significantly impaired.Half of the adults with AMC reported a history of fracture during their lifetime.
A total of 14.3%, 22.5% and 25.0% of patients had lumbar spine, femoral neck and total hip BMD Z scores, respectively, below − 2, i.e. below the expected range for age as determined by the International Society for Clinical Densitometry.Hip BMD in patients with AMC was lower than expected in the healthy population, but this was not the case for the lumbar spine.The level of precision for BMD measurement of the lumbar spine, femoral neck, and total hip is excellent with Lunar iDXA 22 .The BMD values observed in patients with AMC cannot be explained by random measurement variability due to the instrument but indicate a true change in BMD.In our cohort, 30.4% of patients had a Z score less than − 2. This prevalence seems high and significant, especially when compared with the prevalence of low bone mass (defined as a Z score ≤ − 2) in other diseases that affect young people and are associated with an increased risk of fragility fractures, such as cystic fibrosis (52%), Cushing disease (44.5%), human immunodeficiency virus infection (35%) and systemic lupus erythematosus (17.3%) [23][24][25][26][27] .
Hall et al. have argued that fractures occur in patients with AMC not only because of the difficulties of delivery but also because of the BMD deficit associated with AMC 8 .In a study on a pediatric population with AMC, Spencer et al. reported that ambulatory function was weakly correlated with lumbar spine BMD.This observation suggest that, in this population, loading stimulation of the lower limb bones is not sufficient for effective bone remodeling, as also observed in patients with spinal cord injuries 28 .However in our study, patients with limited walking ability in the 6MWT did not have significantly lower BMD.Proteins encoded by TNNI2, TRPV4 and ZC4H2 are expressed by osteoclasts and osteoblasts and are involved in bone remodeling [29][30][31] .Germline mutations in these genes and their effects on protein expression and/or protein function may therefore at least partly explain the bone demineralization observed in patients with AMC linked to these genes.In addition, the fetal hypokinesia observed in patients with AMC may be responsible for the defect in BMD gain during this period, which persists into adulthood 6 .
DXA is the gold standard for measuring BMD to estimate future fracture risk 32 .Cohen et al. demonstrated that a low BMD defined by a Z score of − 2.0 or less in premenopausal women is associated with lower bone stiffness via voxel-based finite element analysis and alteration of the bone microarchitecture, even in the absence of a fragility fracture 33 .Therefore, having a BMD Z score of − 2 or less is likely to mean that the bone is structurally and biomechanically more fragile.The association between BMD on DXA and the risk of future fracture in patients with AMC, as observed in postmenopausal and premenopausal women 27,34,35 , needs to be investigated.In our cohort, half of the adults with AMC experienced a fracture in their lifetime, mainly at a peripheral location.Like Spencer et al. we did not observe an association between lumbar spine or hip BMD and a history of fracture 14 .However, the poor quality of the fracture data collected in this study and in that of Spencer et al. prevents us from drawing any firm conclusions 14 .In addition, the cross-sectional design of our study did not allow us to precisely assess the ability of DXA to predict fracture risk in the AMC.
Reliable measurement of BMD by DXA requires proper positioning of the lumbar spine and femoral necks, making this examination difficult to interpret in patients with structural skeletal abnormalities or in patients with limited joint range of motion 32 .Axial rotation of the vertebrae in patients with scoliosis leads to overestimation of the BMD beyond 5° of rotation 36 .A 10° internal rotation of the leg was reported to artificially increase femoral neck BMD, whereas the opposite effect was observed for external rotation 37 .In our study, 4 patients had severe scoliosis, making the BMD measurements uninterpretable.Eighteen femoral neck measurements were also considered uninterpretable due to incorrect patient positioning.However, all patients had at least one interpretable measurement, as we measured BMD at 3 sites (the lumbar spine, right hip and left hip), indicating that BMD measurement remains feasible in patients with AMC.Careful analysis of DXA results is essential to avoid drawing erroneous conclusions in adults with AMC.BMD measurements should preferably be performed on the hip with a normal range of motion to avoid difficulties in patient positioning, which can lead to measurement errors.
Vitamin D deficiency was observed more frequently in patients with AMC, particularly in patients with limited mobility, than in the general population.Furthermore, in this vitamin D-deficient population, 1 in 2 patients had hypocalcemia, indicating major impairment of phosphate and calcium metabolism, which is known to have a negative impact on bone health.Vitamin D is also important for muscle health.Vitamin D insufficiency is associated with the development of sarcopenia and accelerates the decline in physical performance 38,39 .Muscle weakness has been reported to occur in patients with AMC 11 .In the present study, patients with vitamin D deficiency exhibited weaker performance in the 6MWT.Screening for and correcting vitamin D deficiency, particularly in patients with walking disability, could be a health intervention with beneficial consequences for both bone and muscle function.
We believe that our study is among the largest bone densitometry studies on AMC, and it is the first to assess the prevalence of vitamin D insufficiency in adults with AMC.www.nature.com/scientificreports/However, our study had several limitations.The small sample size and the heterogeneity of the patients, due to the clinical definition of AMC and the rarity of this pathology, did not allow us to draw strong conclusions due to low statistical power.Not all patients underwent DXA analysis and vitamin D assays.In particular, vitamin D and calcium measurements were not systematically included in our screening protocol, which explains the missing data.We cannot exclude the possibility that the incidence of fractures was underestimated due to the recall bias inherent in the study design and the lack of systematic radiographic screening for vertebral fractures, although these fractures are often underdiagnosed.The proportion of fractures caused by low-level trauma equivalent to a fall from standing height or less could not be determined due to the lack of precise data in the medical records.We are therefore unable to estimate the prevalence of fragility fractures.Patients with severe spinal and hip contracture have difficulties positioning the spine and femoral neck as recommended, which can lead to inaccurate BMD measurements.We took this into account by asking two independent readers to assess the quality of the DXA scans.In our cohort, the prescription of vitamin D was not guided by prespecified instructions depending on the patient's pathology.Nevertheless, we observed that patients who received vitamin D had a lower weight.Therefore, we cannot exclude the possible existence of a selection bias in this analysis.Finally, to be included, patients had to visit the French National Center for AMC to obtain an expert evaluation, which could have induced a selection bias.
In conclusion, adults with AMC had lower hip BMD than expected for age and a higher frequency of vitamin D insufficiency.A history of fracture was common in this population.Screening for low BMD by DXA and specific interventions, such as vitamin D supplementation, should be considered in adults with AMC.DXA scans must be interpreted carefully, ensuring that the measurements are not influenced by scoliosis or difficulties in achieving the correct positioning, to avoid drawing erroneous conclusions.

Table 1 .
Characteristics of adult patients with arthrogryposis multiplex congenita.AMC arthrogryposis multiplex congenita, BMI body mass index, ND no data.pvalues:Student'sttest, the Mann-Whitney U test or the chi-square test between patients with Amyoplasia and patients with other types of AMC.Q1: first quartile, Q3: third quartile.Figure 2. Arthrogryposis multiplex congenita and difficulties in measuring bone mineral density with dualenergy X-ray absorptiometry.The dual-energy X-ray absorptiometry results were uninterpretable for some arthrogryposis patients due to severe scoliosis (A) or hip joint contracture with mechanical arthropathy (B).These data were excluded from the analysis.Vol.:(0123456789)ScientificReports | (2024) 14:8206 | https://doi.org/10.1038/s41598-024-58083-xwww.nature.com/scientificreports/lowerthanzero for all patients (p < 0.001) and for both subgroups (Amyoplasia and other types of AMC).The mean femoral neck and total hip BMD did not differ between the Amyoplasia subgroup and the other types of AMC subgroup (Table2).The prevalence of total hip BMD Z scores below -2 was greater in the other types of AMC subgroup than in the Amyoplasia subgroup (7/15, 46.7% vs. 3/27, 11.1%, p = 0.003).A Z score of less than -2 at one site was observed in 30.4% (14/46) of patients with AMC.

Table 2 .
Bone mineral density and vitamin D deficiency in adult patients with arthrogryposis multiplex congenita.AMC Arthrogryposis multiplex congenita.p values: Student's t test or chi-square test between the Amyoplasia group and the other types of AMC group.Bold, significant p value (< 0.05).

Table 3 .
Factors related to lower bone mineral density in patients with AMC.In bold: statistically significant (p value < 0.05).OR odds ratio, CI confidence interval, BMD bone mineral density, BMI body mass index, 25-OHD 25-hydroxyvitamin D, 6MWT 6-min walk test, FIM functional independence measure.